Classical Phenylketonuria (PKU) is a birth defect caused by an inborn error in amino acid metabolism. The genetic disorder is autosomal recessive and is characterized by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) which catalyzes the hydroxylation of phenylalanine to tyrosine. The lack of activity causes elevated phenylalanine level in blood and results in severe mental retardation of the untreated children. The prevalance of PKU among Caucasions is about 1/10,000, and 2% of the population are carriers of PKU. We have previously reported the isolation and characterization of a full-length human PAH cDNA clone which was used to detect restriction fragment- length polymorphisms (RFLP's) at the PAH locus and led to the development of an analytical method for prenatal diagnosis of PKU. This method of analysis however, is applicable only to those families with prior PKU history. RFLP haplotypes for both normal and PKU alleles have been determined for Northern European PKU families. We observed that 90% of mutant alleles are restricted to 4 distinct RFLP haplotypes. Cloning and sequencing analyses of two to these prevalent mutant alleles have revealed point mutations affecting normal mRNA splicing and an amino acid substitution. Oligonucleotides specific for the mutations have been used as hybridization probes to demonstrate that there is a tight association between these mutations and the particular RFLP haplotypes among mutant alleles due to linkage disequilibrium. Additional prevalent PKU genes will be characterized by sequence analysis, and their respective frequencies as well as haplotype associations will be determine. If PKU is indeed caused by a limited number of prevalent mutant alleles, it will be possible to develop a cassett of specific oligonucleotides for carrier detection in the population without prior family history of PKU. Furthermore, mutant alleles in individuals of the less severe hyperphenylalaninemic phenotypes will also be analyzed, which may contain structural mutations that will provide further insight into the structure-function relationship of the enzyme. In addition, the cis-acting elements in the PAH gene responsible for its liver-specific expression and regulation will be characterized and the corresponding trans- acting factors identified. Finally, we will be characterized and the corresponding trans-acting factors identified. Finally, we will attempt to create an animal model of PKU through transgenic mouse technology in order to better understand the pathophysiology of mental retardation in patients.